The long term objectives of this research are to understand the mechanisms by which ion channels gate their pores. Research in this proposal focuses on the large conductance Ca2+ and voltage-activated (BK) channel, which plays a key role in many physiological functions, including control of muscle contraction, regulation of neuronal excitability, and control of transmitter release. BK channels are tetramers, with one voltage sensor, two high affinity Ca2+ sensors, and one low affinity Ca2+ sensor on each of the four subunits. Although much progress has been made towards understanding the contributions of these different sensors in activating the channel, a comprehensive kinetic mechanism to describe gating at the single-channel level is not yet available. To develop this mechanism, channels will be expressed in HEK 293 cells and Xenopus oocytes, and currents will be recorded from single BK channels in excised patches of membrane using the patch clamp technique. The single-channel data will then be analyzed by simultaneously fitting two-dimensional dwell-time distributions of adjacent open and closed interval durations obtained over wide ranges of Ca2+ and voltage to determine the underlying gating mechanism. Aim 1 will isolate and characterize the contribution of each of the three types of Ca2+ sensors to the gating. This will be done in the presence of the voltage sensors to characterize possible interactions among the various sensors. The hypothesis to be tested is that the gating of BK channels modified to have one type of Ca2+ sensor and one voltage sensor per subunit will be described by two-tiered 50 state allosteric gating mechanisms. Aim 2 will use the information obtained in Aim 1 together with additional experimental information to develop a comprehensive kinetic gating mechanism for wild type BK channels with their full complement of one voltage sensor and three different Ca2+ sensors per subunit. The hypothesis to be tested is that the Ca2+ and voltage dependent gating of BK channels is consistent with large two-tiered, recursive, 1250 state allosteric gating mechanisms. The kinetic gating mechanisms to be developed will specify the number of states the channel enters during gating, the transition pathways among the states, the rate constants for the transitions, the voltage and Ca2+ dependence of the rate constants, the allosteric changes in the opening and closings rates for each activated sensor, and the interactions among the various sensors. The ability of the model to describe gating will be tested for single-channel currents and also for published macroscopic ionic and gating currents. Defective BK channels are associated with hypertension, bladder disorder, epilepsy, paroxysmal movement disorder, autism, and mental retardation. The information to be obtained about gating mechanism should be useful towards identifying and understanding disease processes associated with BK channels and designing therapeutic interventions to restore disrupted physiological function. PUBLIC HEALTH RELEVANCE: Large conductance calcium and voltage activated potassium (BK) channels are involved in many key physiological processes including controlling skeletal and smooth muscle contraction, regulating the excitability of nerve cells, and modulating hormone and transmitter release. Defective or missing BK channels have been implicated in hypertension, bladder disorders, epilepsy, paroxysmal movement disorder, autism, and mental retardation. The proposed studies will provide insight into how BK channels function, which will be useful towards understanding disease processes associated with BK channels and in the development of therapeutic interventions to restore disrupted physiological function.